Studies On Thermaltolerance Of Frankliniella Occidentalis,Cloning And Expression Of Heat Shock Protein 70 Genes

Frankliniella occidentalis belongs to Thysanoptera,Thripidae,and Frankliniella.It is an important worldwide insect pest of ornamental plants and economic crops.Against the background of global warming,F.occidentalis has invades and spreaded all over the world quickly.In order to study the effect of temperature on the invasion,distribution and population dynamics of F.occidentalis,we compared and analyzed temperature tolerance of F.occidentalis before and after long-term feeding under laboratory conditions when they were be traeated by cold and heat stress.High-throughout sequencing were applied to obtain the transcriptome information of F.occidentalis under different temperature stress.We obtained HSP70(Heat Shock Protein 70,HSP70)genes from transcriptome and cloned and analyzed their expression patterns.Analysing the tolerance of F.occidentalis under temperature stress at the ecological and molecular level can provide a theoretical basis for its diffusion and adaptation mechanisms,and provide guidance for the future forecast distribution and comprehensive management.The main results are as follows:1.We analyzed the change of thermotolerance of reared population which was at a constant temperature(26±1?)for a long time by comparing the survival rate of different sexes and stages of F.occidentalis after they were exposed to both high and low extreme temperatures for an hour.Results indicated that the thermotolerance of females,males and second instar larvae were all improved after they were reared at long-term constant temperature in laboratory.When exposed to cold stress,the initial lethal cold temperature of adults and larvae reduced from-8?to-11?,and the LT50(the temperature leaded to 50 percent mortality)of females,males and larvae reduced from-12.5,-11.5 and-12.8? to-13.2,-13.8 and-13.3 ?,respectively.When exposed to heat stress,the initial lethal temperatures of adults and larvae increased from the initial 33? to 37?,and the LT50 high temperatures of females,males,and larvae increased by 1.1,2.8 and 3.3?,respectively.2.Transcriptome sequencing was performed on Illumina sequencing platforms for F.occidentalis at low temperature(-13?),high temperature(40?)and normal temperature(26?).A total of 79,516 unigenens were obtained by assembling and splicing.Compared with the Nr(Non-redundant)database,28,765 annotated genes were received successfully.Compared differential gene among the three transcriptome samples,there were 134 unigenes expressed differentially between-13 and 26?,among these were 60 up-regulated unigenes and 74 down-regulated unigenes;there were 333 unigenes expressed differentially between 40 and 26?,among these were 153 up-regulated unigenes and 180 down-regulated unigenes;there were 230 unigenes expressed differentially between 40 and-13?,among these were 162 up-regulated unigenes and 68 down-regulated unigenes.3.Based on HSP70s unigene fragment information in the transcriptome database,three full-length Fohsp70s contained open reading frame(Opening Reading Frame,ORF)were cloned and obtained by RT-PCR(Real-time PCR)and RACE(Rapid-amplification of cDNA ends).The three cDNA sequences were named Fohsp703(KY914546),Fohsc704(KY914547)and Fohsc705(MF377626),respectively.The complete cDNA of three HSP70 genes are 2 106,3048 and 1 917 bp,and their ORF are 2,040,2,073 and 1,476 bp,which encode 679,690 and 491 amino acids,respectively.Be different from the reported FoHSP70s,the three FoHSP70s ended with APAA,EKKN and GIFL.Sequence analysis suggested that FoHSP704 and FoHSP705 shared high similarity with HSP70s of other species,but FoHSP703 did not.At the same time,phylogenetic analysis showed that the three HSP70s were distributed on four different branches with the previously studied FoHSP70.Compared the genomic sequence with the cDNA sequence,we found 0,4 and 6 introns in Fohsp703,Fohsc704 and Fohsc705,respectively.4.The expression patterns of Fohsp703 and Fohsc704 of different sexes and development stages under cold and heat stress were studied by real-time quantitative PCR.The results showed that the expression of the two Fohsp70s could be induced by high and low temperature stress,but the expression patterns were different.When exposed to low temperature stress,the expression levels of Fohsp703 and Fohsc704 were the highest in females compared to males,pupae and larvae,especially at-10,-12 and-14?.Under high temperature stress,the expression level of Fohsp703 was the highest when second instar larvae were at 37 and 39?.There was no significant difference in the expression level of Fohsc704 in the adults,but the expression level increased when the pupae were at 35? and the larvae were at 370C.